Chinese hamster ovary cell line transfected with 30K gene
a technology of hamster ovary and cell line, applied in the field of cho (chinese hamster ovary) cell line transfected with 30 k gene, can solve the problems of affecting the stability of target, reducing survival rate, and lowering the productivity of target proteins
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example 1
Cell Line and Culture Condition
[0044] The recombinant Chinese Hamster ovary (CHO) cell lines producing human erythropoietin (EPO) and 30 K protein originating from silkworm hemolymph were grown in DMEM / F-12 (1:1) (JRH Bioscience) supplemented with 10% fetal bovine serum (FBS, Gibco), L-glutamine, 15 mM HEPES buffer, and penicillin / streptomycin (Gibco). Cells were Captained at 37° C. in a humidified air atmosphere with 5% CO2. To produce recombinant human EPO, the growth medium with 10% FBS was replaced with serum-fee medium, EXCELL 301 (JRH Bioscience).
example 2
Establishment of Stable Recombinant CHO Cell Line Producing 30Kc6
[0045] A cDNA clone containing 30Kc6 was kindly provided by S. Izumi (Department of Biology, Tokyo Metropolitan University). He indicated that it was constructed as follows (personal communication): A DNA fragment for one component of 30 K proteins (30Kc6 GenBank accession number, X07552) was amplified by RT-PCR from fifth larval fat body RNA with synthetic oligonucleotide primers specifically used for 30Kc6.
[0046] The resulting DNA fragments were digested by EcoRI and inserted into the EcoRI site of pBluescript KS+. We cloned the entire open reading frame of the 30Kc6 into the mammalian expression vector pcDNA3 (Invitrogen).
[0047] The pcDNA3 / 30Kc6 or pcDNA3, the vector alone as a control, was transfected to CHO cells by the LipofectAMINE Reagent (Gibco) according to the manufacturer's instructions. For the establishment of stable cell lines expressing 30 K protein, CHO cells were transfected with the indicated pla...
example 3
RT-PCR
[0050] The total cellular RNA was extracted by RNA isolation kit PURESCRIPT (Gentra systems) according to the manufacturer's instructions. RNA concentration was measured spectrophotometrically. Using a sequence specific primer (30Kc6 reverse primer: 5-TCG TTT TCA GCT TCA GCT TTA-3), cDNA was synthesized from 3 μg of total RNA.
[0051] PCR was performed for 36 cycles by the following program for each cycle: denaturation at 95° C. for 1 min. annealing at 60° C. for 30 s, and extension at 72° C. for 1 min using a 30Kc6 forward primer (5-ACA GTG TTG TGA CTG cTT TCA-3) and reverse primer (5-TCG TTT TCA GCT TCA GCT TTA-3). The PCR product was analyzed on 1% agarose gel by electrophoresis.
[0052]FIG. 1 is the photograph of RT-PCR analysis of 30 K mRNA in the stable CHO cell times transfected with 30 K expression construct. The expected size for the PCR product is 890 bp for the 30 K protein. Lane M, 1 kb molecular weight ladder; lane 1, transfected with pcDNA3; lane 2, transfected w...
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